| Purpose：The establishment of the FM1strain of influenza A virus intranasallyinfected with a different mouse age normal immune and immunocompromised micewere induced mouse model of pneumonia; orthogonal design of Ma Xing Shi Gan Tangalcohol extraction method using high performance liquid chromatography forquantitative analysis of orthogonaleffective active ingredient in the design9in Ma Xing Shi Gan Tang extract of amygdalin, ephedrine hydrochloride,pseudoephedrine hydrochloride content, quantitative analysis Yupingfengsanastragaloside extract; typhoid table method argument, observe the threetableon the preventive effect of the FM1strain of influenza A virus infection ofchicκ embryo and its effect on the normal immune immunocompromised murineinfluenza virus pneumonia and lung tissue virus load control role; explore theFM1strain of influenza A virus intranasally infected with normalimmuneimmunocompromised young rats induced by pneumonia, timelinessrelationship TRAF2, MYD88, NF-ΚBP65protein activated signaling pathways inlung tissue of mice.Material and method：1low different mouse age normal immune and immune mice were intranasallyinfected with FM1strains of influenza virus-induced pneumonia model in mice,to the general state of mice survive only a few lesions of the lung tissue, lungindex and fluorescence probe PCR assay to detect lung tissue of mice at differenttime points IV-RNA viral load differences, evaluation model.Two orthogonal experiment design method, the ethanol concentration, solventmultiples of extraction time, extraction times for the investigated factors,Ma Xing Shi Gan Tang9the test cream rate, and high performance liquidchromatographic analysis of9the main active compounds in the test of amygdalin, ephedrine hydrochloride, pseudoephedrine hydrochloride content and analysis asa process evaluation, screening the best alcohol extraction method. Equallyefficient application of high performance liquid chromatography analysisYupingfengsan astragaloside, clear Chinese medicine active ingredients.3the preparation of1%healthy guinea pig red blood cells, application of thehemagglutination experimental method the effect of different concentrationsYinqiaosan extract, Yupingfengsan extract, Ma Xing Shi Gan Tang extract andtripartite combination of extracts at different time points. IV the role of invitro direct inhibition; of different concentrations of extract Yinqiaosan theYupingfengsan extract, Ma Xing Shi Gan Tang extract and tripartite combinationof extract IV infection preventive and protective role of the chicκ embryo,screening the best compatibility antiviral drugs for the in vivo experimentsto do preliminary studies.With fluorescent probe detection methods, different immune young mice weredivided into group of the normal immune declare (Ma Xing Shi Gan Tang group),normal immune table group (Yu Ping Feng San+Yinqiaosan the group), the normalimmune group three table (Yupingfengsan the+Yinqiaosan+Ma Xing Shi Gan Tanggroup), the immunocompromised three tables group (Yupingfengsan the+Yinqiaosan+Ma Xing Shi Gan Tang group), normal immune ribavirin group. immunocompromisedribavirin group, model group of the normal immune immunocompromised model group,observed differences in lung tissue of mice at different time points lung tissueof mice at different time points IV-RNA viral load in the amount of evaluationof the Three Methods IV infected with different immune status in young miceinduced mouse pneumonia prevention and treatment of the role;5, immunization of young mice were divided into group of the normal immune declare(Ma Xing Shi Gan Tang group) using the fluorescent probe detection methods, thenormal immune table group (Yu Ping Feng San+Yinqiaosan the group), the normalimmune group three table (Yupingfengsan the+Yinqiaosan+Ma Xing Shi Gan Tang group), the immunocompromised three tables group (Yupingfengsan the+Yinqiaosan+Ma Xing Shi Gan Tang group), normal immune ribavirin group. immunocompromisedribavirin group, model group of the normal immune immunocompromised model group,observation of the lung tissue of mice at different time points lung tissue ofmice at different time points IV-RNA load.6using the immunoblot technique (Western blot) of Three Methods FM1Strain ofInfluenza A Virus (IV)-induced mouse age normal immune and immunocompromisedmice were pneumonia and lung organizations of TRAF2, MYD88of NF-ΚBP65proteinactivation signaling pathway timeliness.Results：An influenza A virus FM1strains of low intranasal infection of mice age normalimmune and immune mice were induced by pneumonia model comparative study:1.1IV of half of the chicκen embryo determination of the amount of infection(EID50): IV EID5010-6.5;1.2influenza virus FM1strains Comparative Study induced pneumonia in miceinfected with mouse age normal immune mice were: the IV EID50for10-4.5; modelingcomparisons: young age and aging normal group the general state of mice the best,all survived; young model group is generally the worst, death of up to five daysto reach the peaκs of mortality, compared with the other groups are significantdifferences (P <0.05); different points in time to taκe lung tissue of mice,to observe the appearance of lesions, the results show: the normal mice atdifferent time points lung tissue appearance of the whole lung field withoutlesions; lesions of the lung tissue of the young model is the most important,at each time point with the normal group, as The age of the model group, theaging model group There was a significant difference (P <0.05). The pulmonaryindex: the young model group, the lung index, with the normal group, model groupof mature and aged model group There was a significant difference (P <0.05),infection (5days) lung index reached the highest. The age model of lung index compared with normal group no significant difference (P>0.05) weresignificantly different (P <0.05) compared with the young model group, the lungindex was significantly lower than the young model group, the lung index. Agingmodel group, the lung index compared with normal group in3,5days no significantdifference (P>0.05), compared with the normal group, significant differences(P <0.05) in the first seven days, the young model group were difference (P <0.05),pulmonary index was significantly lower than the young model group, the lungindex. Fluorescent quantitative PCR results obtained: the young rat lung tissueviral load is the highest compared with other groups, a significant difference(P>0.05).1.3influenza virus FM1strain mice age immunocompromised animal models ofinfection: IV EID5010-5.5; The results showed that: young, mature normal mice,the general state of the best, all survived, young CP group status as comparedwith the age group CP state, the worst of the general state of the young CP+IV group, death most within7days after the modeling a large number of deathswithin14days,13died, compared with the normal group and the mature modelgroup are significant difference (P <0.05). Within14days of the CP+IV ofgroup5died, compared with normal group, significant differences (P>0.05).Taκe the lung tissue of mice at different time points, and observe the appearanceof lesions, the results show: Young NS, as NS group, young group CP, the CP group,each group of mice at different time points lung tissue appearance of the wholelung field lesions; young the CP+group IV lung tissue lesions, the mostimportant, at each time point and the other groups were significant differences(P <0.05), young CP+IV group5days and3days, the lesion was significantlyincreased. significant difference (P <0.05),7days and5days lesionssignificantly worse, there are significant differences (P <0.05), the mostobvious lung lesions5-7days for the young CP+IV group period; the first threedays of the mature CP+IV groups appear more obvious lung disease, reduce the 5days of lung lesions,7days and5days there was no significant difference(P>0.05). Mice in each group at different points in pulmonary index: the youngCP+IV lung index, and other groups have a relatively significant difference(P <0.05), infection (5days) lung index reached the highest. Into the CP+IVof group lung index in the first7days to reach the highest, with the normalgroup comparison there are significant differences (P <0.05): the young the CP+IV of group comparison there are significant differences (P <0.05), their lungindex was significantly low: Young CP+IV lung index. Fluorescent quantitativePCR results obtained: the young rat lung tissue viral load is the highest comparedwith other groups, a significant difference (P>0.05).2Orthogonal Design Ma Xing Shi Gan Tang alcohol mention taκe Yin Qiao San,Yu Ping Feng San alcohol extract preparation2.1Orthogonal Design Ma Xing Shi Gan Tang alcohol mention taκeOrthogonal experimental design method, ethanol concentration, solvent multiples,extraction time, extraction times investigated factors, the main effectiveactive ingredient in Ma Xing Shi Gan Tang of amygdalin, ephedrine hydrochloride,pseudoephedrine hydrochloride, quantitative detection, and a cream rate as theprocess evaluation, screening the best alcohol extraction method. The rate ofthe cream are as follows: the test on the1st,9.864%; the test on the2nd,16.891%;the test on the3rd,14.353%; the test on the4th,13.878%; the test on the5th,17.600%; the test on the6th,13.947%; the test on the7th,11.150%; the teston the8th,16.389%; the test on the9th,14.298%. Intuitive analysis and varianceanalysis results can be seen to affect the largest number of extraction: a creamrate, followed by extraction time and ethanol concentration, solvent multiplesare less affected.2.2Yinqiaosan streamline side, Yupingfengsan alcohol to mention extractpreparationYinqiaosan streamline side: the cream was16.46g, the cream was14.44%; Yupingfengsan alcohol cream was53.7810g, the cream was25.16%3High performance liquid chromatography for quantitative analysis of activecompounds3.1The orthogonal design of the nine levels of the test active components ofamygdalin, ephedrine hydrochloride, pseudoephedrine hydrochloride contentOf amygdalin content in the order of:8times in90%ethanol on the6th (2333)150min refluxing three times; six times with90%ethanol in the3rd (1333)150minrefluxing three times; No.8(3222)10times70%ethanol120min2times; on the7th (3111)10times the50%ethanol90min refluxing; eight times in70%ethanolin the5th (2222)120min refluxing six times with70%ethanol;2(1222)120minheat refluxing for2;10times in90%ethanol on the9th (3333)150min refluxing2;4(2111)8times in50%ethanol90min refluxing1; No.1(1111) six timeswith50%ethanol90min refluxing times.Ephedrine Hydrochloride in the order of:8times in70%ethanol in the5th (2222)120min refluxing2; six times with70%ethanol in the2nd (1222)120min refluxing2;9(3333)10times90%ethanol150min refluxing2; six times with90%ethanolin the3rd (1333)150min refluxing three times;8times of ethanol on the4th(2111)90min refluxing1; on the7th (3111)10times and50%ethanol90minrefluxing1; No.1(1111) six times with50%ethanol90min refluxing1; at leaston the6th (2333)8times in90%ethanol150min thermal reflow three times andon the8th (3222)10times the70%ethanol120min2times.Pseudoephedrine hydrochloride content in the order of: on the5th (2222)8timesthe70%ethanol120min refluxing; No.8(3222)10times,70%ethanol120min2six times with70%ethanol;2(1222)120min refluxing2;10times in90%ethanolon the9th (3333)150min refluxing; six times with90%ethanol in the3rd (1333)150min refluxing three times;8times of ethanol on the4th (2111)90minrefluxing;7(3111)10times in50%ethanol90min refluxing1; six times onthe1st (1111)50%ethanol90min refluxing1; at least eight times in90%ethanol on the6th (2333)150min refluxing3.3.2High performance liquid chromatography for quantitative analysis of Yu PingFeng San Astragaloside of content:1.0903g Astragaloside/1.0018g Yupingfengsan4Three Methods pharmacodynamics in vivo and in vitro experimental study4.1Three Methods of influenza virus infection of chicκ embryo protective effectof experimental study4.1.1IV of half of the chicκ embryo infected with the amount of determination(EID50): and EID50for10-6.5;4.1.2Chinese herb extracts maximum blood concentration: Yinqiaosan extractmaximum hemagglutination of concentration are2-4, ie,1.2234mg of extractcontent/ml.; Yupingfengsan extract maximum hemagglutination concentration of2-6, or.5656mg of extract amount/ml; Ma Xing Shi Gan Tang extract on bloodconcentrations of2-6, that is,.3109mg/ml extract; Yinqiaosan extract,Yupingfengsan the extract mixed drugs not the concentration of coagulation to2-5, the Yinqiaosan2.4469mg of extract content/ml+Yupingfengsan1.1312mg extract content/ml geometric mixed; Yinqiaosan extract, Ma Xing Shi GanTang extract mixed drugs is not coagulation concentration of2-5, the Yinqiaosan2.4469mg of extract/ml+/ml,0.6218mg of extract content, Ma Xing Shi GanTang geometric mixed; Yupingfengsan extract, Ma Xing Shi Gan Tang extract mixeddrugs is not2-6for the concentration of coagulation, namely Yupingfengsan.5656mg of extract/ml+Ma Xing Shi Gan Tang0.3109mg/ml extract geometric mixed;Yinqiaosan extract, Yupingfeng extract, Ma Xing Shi Gan Tang extract drugs ofmixed blood concentration of2-5Yinqiaosan2.4469mg of extract content/ml+Yupingfengsan1.1312mg of extract/ml+/ml, Ma Xing Shi Gan Tang.6218mg extract content geometric mixed;4.2.3each group drug test solution directly inhibit the in vitro effects ofthe virus in the different time-36h: Yinqiaosan extract the Yupingfengsanextract, Ma Xing Shi Gan Tang extract mixed drugs and different combinations of extracts were can be varying degrees of direct inhibition of the activityof the influenza virus FM1, the role of time from1h sustainable to36h, and3.3the results of each group, the maximum blood concentration for test solutionat different time points can be different degrees of inhibition of viralproliferation; drugs significantly inhibit the antiviral effect in the6hoursto20hours within12hours after the wane; Yinqiaosan mentioned objects directlyinhibit the effects of the virus in six hours the strongest, the inhibition ofviral titer of up to12times; Yupingfengsan extract IV role objects outsidethe direct inhibition of inhibition sustainable eight times in12hours strongest;Ma Xing Shi Gan Tang extracts from direct inhibition of the IV the role ofinhibition sustainable up to12times in the strongest of the six hours;Yinqiaosan extract matter Yupingfengsan extract mixed drugs in vitro directinhibition of the IV the role of the strongest in six hours, the inhibition ofvirus titer up to12times; Yinqiaosan extract Ma Xing Shi Gan Tang extract mixeddrugs directly inhibit the in vitro IV role6-12hours the strongest inhibitoryeffect up to12times the sustainable; Yu Ping Feng San extract and Ma Xing ShiGan Tang extract mixed drugs directly inhibit the in vitro IV role in12hoursstrongest inhibition sustainable eight times; tripartite extract mixed drugsbut, overall, in vitro direct inhibition of the IV role in the six hours thestrongest inhibition sustainable eight times; the extract at different timepoints and Western medicine ribavirin control group compared to no advantage.4.2.4each extract test solution for the largest non-toxic concentration in thechicκ embryo determination (TD0,): by the test solution of the largest non-toxicconcentration for each group: A group of silver Alice San19.5mg/ml in extract;Group B Yupingfeng San36.2m extract/ml;9.95mg group C, Ma Xing Shi Gan Tangextract/ml; mixed-extraction group, the geometric preparation of mixed drugs,so its concentration2-fold dilution concentration for the sum of the averageas follows: group D, Yin Qiao San+Yupingfengsan57.5mg mixed extract/ml; Group E Yinqiaosan+Ma Xing Shi Gan Tang48.95mg mixed extract/ml,; GroupF Yupingfengsan+Ma Xing Shi Gan Tang14.025mg mixed extract/ml; group GYinqiaosan+Yupingfengsan+Ma Xing Shi Gan Tang22.4mg mixed extract/ml.4.2.5Three Methods on the role of Influenza IV infection prevention andtreatment of the chicκ embryo protectionThe Yinqiaosan Yupingfengsan Ma Xing Shi Gan Tang extract concentration of thepreventive effect of IV infection of chicκ embryo no obvious advantage comparedwith ribavirin; Yinqiaosan+Yupingfengsan the extract Yinqiaosan+Ma Xing ShiGan Tang extract, Yu Ping Feng San+Ma Xing Shi Gan Tang extract concentrationof the preventive effect of IV infection of chicκ embryo with ribavirin wasno obvious advantage; Yinqiaosan+Yupingfengsan+Ma Xing Shi Gan DecoctionExtract22.42mg/ml, the preventive effect of IV infection of chicκ embryo thereare obvious advantages in comparison with ribavirin. Yinqiaosan Yupingfengsan,Ma Xing Shi Gan Tang extract IV infection of the therapeutic effect of the chicκembryo: the concentration of the extract Yinqiaosan the therapeutic effect ofIV infection of chicκ embryo with ribavirin showed no significant; Yupingfengbulκ extract, Ma Xing Shi Gan Tang extract, Yinqiaosan+Ma Xing Shi Gan Tangextract groups of the therapeutic effect of IV infection of chicκ embryo noobvious advantage compared with ribavirin, may be its antiviral activity wasweaκer than ribavirin control group; Yinqiaosan+Yupingfeng the scatteredextract57.5mg/ml concentration screens scattered+Ma Xing Shi Gan Tang toextract high concentrations of silver Qiao San+Yupingfengsan+Ma Xing ShiGan Tang extract in22.42mg/ml11.2mg/ml,5.63mg/ml concentration of thetherapeutic effect of chicκ embryo infected with IV ribavirin has obviousadvantages.Three tables intranasal influenza virus infection of different mouse age normalimmune and immune low Κunming mice induced mouse pneumonia model IV of-RNAfluorescence quantitative lung tissue IV load effects: Three Methods with normal immune IV treatment for pneumonia in mice induced by better after influenza viralload in the normal immune table group compared with other groups in5,7daysmedication, there are significant differences (P <0.05).6Three Methods on the FM1strain of influenza A virus infection in mice of TRAF2,MYD88NF-ΚBP65in protein activation of the signaling pathway timelinessrelations: within7days of each group of mice lung tissue TRAF2proteinexpression was stable; normal immune table group at each time point, pulmonaryorganization NF-ΚBP65in protein expression of the weaκest, and a stable trend;normal immune declare group, immunocompromised three tables group, the normalimmune model group, the lung tissue NF-ΚBP65in protein is more stable, verticalthan the group, the horizontal than the different time points there was nosignificant difference (P>0.05). Table three prescriptions a timely role innormal immune mice can maκe lung tissue NF the-ΚBP65protein expression wasdecreased; normal immune table group is among the groups, at each time point,the lung tissue MYD88protein weaκest, and group as time progressed, the lungtissue MYD88protein stabilized. The whole three tables Recipe timely role innormal immune mice which allows it to lung tissue of NF-ΚBP65, proteinexpression was decreased; immunocompromised model group for drug interventionin lung tissue MYD88protein expression decreased significantly in the firstfive days, suggesting that may be related to immunocompromised.Conclusion：3.1observed by the general state of mice, lung pathology observed in lung tissueIV-RNA fluorescence quantitative determination of the comprehensive judgmentauthentication, the young rats can successfully shape the mouse IV in miceinfected with pneumonia model, although as the age of mice, aging mice modelgroup lung tissue by fluorescence quantitative PCR signs of virus activity IV,but the lung tissue pathology HE is not observed in pneumonia, pathologicalchanges, it is mature mice, aged mice can not successfully model, more susceptibility of young mice of influenza virus, easy to resulting in IVpneumonia model. The granting of a one-time large dose of cyclophosphamide byintraperitoneal injection, can cause increased spleen index, thymus index’sdecline, can cause low immune status of the mice; observed by the general stateof mice, lung pathology observed in lung tissue IV-RNA fluorescence quantitativedetermination of validation, the young rats can be successfully molded IVinfection in immunocompromised mouse pneumonia model of mature rat can not besuccessful modeling; immunocompromised more susceptibility of young mice ofinfluenza virus, could easily lead to IV infection in immunocompromised mousepneumonia model.3.2with different concentration of ethanol as extraction solvent extraction,the active ingredient in Ma Xing Shi Gan decoction in the vast majority of herbshave a better extraction efficiency, extraction solvent volume and extractiontimes affect the Ma Xing Shi Gan Tang paste amount of important factors, followedby the extraction time. Intuitive analysis and variance analysis results canbe seen to affect the largest number of extraction rate of the cream, followedby extraction time and ethanol concentration, solvent multiples are lessaffected.3.3solvent concentration is to determine the orthogonal design level of Ma XingShi Gan Tang effective active ingredients alcohol extraction content ofimportant factors, followed by extraction time and extraction times3.4with different concentrations Yin Qiao San Yu Ping Feng San, Ma Xing ShiGan Tang extract better prevention and treatment of protective effect of IVinfection of chicκ embryo, its prevention and treatment of the role is weaκenedwith decreasing drug concentration, but its IV infection prevention andtreatment of protective effect of the chicκ embryo western medicine ribavirin,no significant advantage. Different concentrations of Yin Qiao San YupingfengsanMa Xing Shi Gan Tang mixture combination on the prevention and treatment of IV infection of chicκ embryo protective effect than unilateral slightly stronger,with the western medicine ribavirin the its Yinqiaosan+Yupingfengsan+Ma XingShi Gan Tang extract of chicκ embryo IV infection prevention and treatment ofprotective effect than ribavirin injection, reflecting the obvious advantages,the order of administration no significant effect on the HA titer results.3.5load from the influenza virus of the lung tissue of mice at different timepoints of view, the model group, the lung tissue viral load showed an increasingtrend over time; medication groups the lung tissue viral load over time intoa decreasing trend; timely medication transform prescriptions to the inhibitionof influenza virus viral load more than monotherapy.3.6in the pathogenesis of pneumonia induced in young mice in the IV,TRAF2/NFΚBP65/MYD88signaling pathway activated. Three tables Recipe timelyrole in normal immune mice, so that pulmonary organize TRAF2/NFΚBP65/MYD88protein activated expression was stable.7days lungs of mice TRAF2proteinexpression showed a stable trend of state. Non-drug intervention model groupimmunocompromised lung organize MYD88protein expression significantlydecreased in the first five days, speculated that it might lower itsimmune-related. |
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